It is estimated that in the United States 150,000 stroke-related deaths occur each year, making stroke the country's third-leading cause of death. In nonfatal strokes, ischemic injury to the hippocampus, a brain region known to be selectively vulnerable to ischemia, results in cognitive and memory impairment. After cerebral ischemia, surviving neurons undergo a variety of cellular responses that are not completely understood. The long-term goal of this research is to elucidate mechanisms of responses to cerebral ischemia. The present proposal focuses on one aspect of ischemia, hypoxia, and its effect on the fimctional expression of muscarinic acetylcholine receptors (mAChRs), which are thought to play a role in learning and memory. Our experiments will test the hypothesis that transient hypoxia causes a decrease in functional responsiveness of the m2 mAChRs and that the mechanism involved includes both pre- and post-translational modifications. Experiments are designed to determine the underlying mechanisms that mediate internalization, desensitization, and down-regulation of m2 mAChRs by transient hypoxia. The proposed study will use cultures of enriched primary hippocampal neurons and human embryonic kidney cells and mouse embryonic fibroblasts that stably express wild-type or mutated forms of the m2 mAChR. In Specific Aim 1, experiments will be performed to test the hypothesis that hypoxic insult induces phosphorylation of the m2 mAChR, thereby leading to internalization and decreased responsiveness to muscarinic agonists. In Specific Aim 2, experiments will be conducted to test the hypothesis that desensitization of the m2 mAChR by transient hypoxia is independent of m2 receptor internalization. In Specific Aim 3, experiments will be carried out to test the hypothesis that transient hypoxia decreases the expression of the m2 mAChR mRNA and/or protein. Together, these experiments will determine how hypoxia induces alterations in mAChR signaling and advance understanding of the consequences of stroke and transient ischemic attacks. Results from these studies will be useful in developing therapeutic interventions to reduce the impairment of learning and memory after ischemic insults.